JPS58159420A - Purification method of interleukin 2 - Google Patents

Abstract

PURPOSE:To purify the titled compound, by bringing a solution containing interleukin 2 into contact with porous glass beads, adsorbing the interleukin 2 in the glass beads, and eliminating and eluting the interleukin 2 from the porous glass beads. CONSTITUTION:A solution containing interleukin 2 (IL-2), e.g. a human peripheral blood, splenic cell or human T leukocytic cell, at 5-10pH is brought into contact with porous glass beads (CPG), and adsorbed therein. The ionic strength of the IL-2 solution in the adsorption is preferably <=0.5M based on the NaCl concentration. The adsorbed interleukin 2 adsorbed in the CPG is then eluted with an eluent, e.g. a buffer solution containing 1-99% ethylene glycol or glycerol, and the resultant IL-2 solution is then separated by the concentration with a membrane, salting out, etc. and used for an antitumor drug or diagnostic agent, etc. EFFECT:An economic method having a high recovery ratio, simplified desalting steps and high efficiency.

Description

Detailed Description of the Invention Interleugin 2 (hereinafter abbreviated as l-I L-2-1) is an immune mediating factor that is present in humans, mice, etc. ) 7. The usefulness of killer T cell proliferation and IL-2 is widely recognized.

The crude I L-2 obtained by straining in this way is generally
Because the concentration of IL-2 is low and contains many impurities derived from other IL-2 cells, culture medium, and additives, IL-2 It can be said that refining is essential.

Several methods for purifying IL-2 have been reported.

Gallo et al. of the NIH in the United States salted out IL-2 produced by lymphocytes in human peripheral blood and subjected it to ion exchange chromatography (DEAE-Sepharose), followed by Tris-HCl containing 0.1% polyethylene glycol. Buff fur (0,1M,
Gel filtration chromatography (pH 8,0)
It was purified from Rogel ACA54). They added polyethylene glycol to the buffer solution as a stabilizer when using a gel because IL-2, which contains a small amount of protein, is easily deactivated by adsorption to columns, resins, etc. ) Culture solution? This λ is 1 and the purity is 400 times l L
-2 was obtained with a recovery rate of only 9%. (R, C, G
a1lo cl al-+Proc, Na11.
Acod, Sci, USA, 77,
6134 (+980) However, this method requires one ion exchange resin treatment at the beginning of purification, so it requires desalting operations such as dialysis first.
Because it is carried out on a large scale, this method is not necessarily suitable due to its operability, and the recovery rate of the obtained 1 L -2 is also low. In addition, electrophoresis 5DS-1) Δ〇 li
However, it is extremely difficult to remove the surfactant, thorium dodenyl sulfonate (SDS) in Zasobl, making it unsuitable for clinical use.

Also, Gillis et al.

J, Immunol, ±24(4)
1954 (1980)) and Reich et al. (E, Re1
ch elal, + J, Exp.

Med, 154, 422 (1981)) also uses similar methods for purifying human, mouse, and rat IL-2, such as β-spectral filtration, ion exchange, and various electrophoresis, but they have the same drawbacks. However, it is not suitable for large-scale preparation.

In addition, Brown et al.
noragi-cal Method 33, 3
37-350 (1980)) also purified L-2 produced from mouse and rat spleens by salting out and gel filtration, but the degree of purification was low ((100 times),
Furthermore, when carried out on a large scale, there are drawbacks such as the need for too large a volume of chromatograph gel.

The inventors of the present invention have conducted intensive research to develop the large 1-Wji manufacturing method of 11.
) as an adsorbent, (1) the recovery rate of IL-2 is high, Q) the desalting process can be simplified, (3) the amount of liquid to be handled does not increase because only a small amount of adsorption carrier is needed, etc. They discovered that this is a highly economical and efficient method, and completed the present invention.

According to the method of the present invention, porous glass beads (hereinafter referred to as "C
It is sometimes abbreviated as PG. ) may be used alone. Furthermore, CPG may be used in combination with other biopolymer purification methods, such as ion exchange, gel filtration, chromatography using hydrophobic resins, or electrophoresis, if necessary. like this? Is it easy to rub? This highly purified IL-2 may be derived from horses, rats, or mice, and is naturally applicable to IL-2 culture supernatants produced by genetic engineering techniques. The IL-2 production medium may be a serum medium or a bloodless 7N medium.

Details of the purification according to the present invention will be described below.

First, an IL-2 solution containing contaminants such as proteins was prepared at pH 5.
~10, preferably at pH 6.6 to 8.5 to adsorb IL-2 onto porous glass beads. This solution can be a culture solution, a concentrated solution with membrane properties, salting out, etc.
The resulting protein precipitate may be redissolved or a crudely purified IL-2 solution may be used.

Note that the ionic strength of the l L -2 solution during adsorption does not have a large effect on adsorbed P, but for NaCl
If F exceeds 1.0 M at a degree, the amount of non-adsorbed II-2 will increase, so it is desirable to set it at 0.5 MM or more.

IL-2 thus e-filtered and adsorbed is, for example,
It is eluted with the following eluent. That is, (I) a buffer containing 1-99% of ethylene glycol, glycerin, etc., preferably 20-99%. Note that the buffer here may be any commonly used buffer as long as it is in the region where p i (I l--2 is stable. (2) (i7゜The pH of V-phosphoric acid is 1.
0 to 5.0, preferably 1.5 to 3.5.

Any acid may be used as the eluate as long as its pH is within this range. Also, (3)-10-v
+ CNS +I +Br +ClO4+I-+
A solution of salts such as +Ca + argylammonium ions or a buffer solution containing these salts may be used. In this case, the concentration of salts is 0.1 M or more.
Preferably 6- If the distance is 0.4 to 1.0M.

The bathing method itself may be a normal method, and it can be done by washing the CPG adsorbed with IL-2 with the above eluent1.The IL-2 solution obtained by the above steps is as follows: The IL-2 can be used as it is or after being fractionated by membrane concentration, salting out, etc., for appropriate IL-2 applications. Alternatively, as desired, such IL-2 solution may be used as it is or after a similar treatment, and then subjected to further chromatography treatment using one of various types of trees to improve its purity. You can do one more thing.

In the case of gel treatment, the chromatography resin used in combination with CPG treatment may be any resin with a molecular weight fraction ranging from several thousand to about 1,000 yen, and in the case of ion exchange resins. , diethylaminoethyl (D
EAE) Anion exchange systems such as cellulose may also be used.

The CPG treatment of the present invention may also be combined with column chromatography or electrophoresis using a hydrophobic ion exchange resin such as phenyl sepharose.

In particular, the M4 manufacturing method, which is a combination of CPGr's Calano lithography and gel filtration column chromatography (either column chromatography can be performed one time first), produces high-purity products. l L-2 can be changed. In addition, CPGt Koyoru no J in non-inventive method 1
Chromatography Q: It is best to use the same technique as in general column chromatography 13, except that CPG is used as the adsorbent.

Incidentally, for example, in the case of human IL-2, in the example performed with this combination, the purified product obtained had a concentration of 5XIO' to 106 u n as measured by the method of Gillis et al. described below.
It showed an activity of 1t/my and electrophoresis (5DS-PA
It is a protein with a molecular weight of +5,000 Daltons and a molecular weight of 25. It didn't contain anything bigger than OOO Dalton.

The activity of IL-2 was measured using the method of Kiris et al. (S, G
11lis et al, +J, Exp, Me
d, +152 +1709 (1980))t.

1. Concentrate s o o untt/++l) using Holofiber HIP5 (manufactured by Amicon) and reduce the volume to 4
After adjusting to 00rxl, solid ammonium sulfate 2401 was added and the mixture was strained to 85% of the saturated ammonium sulfate concentration to precipitate proteins including TL-2. Then centrifugation (6,00 rpm for 30 minutes)
The protein was thus separated.

The separated protein was dissolved in 266 ml of a 0.1 M (tris) hydroxyaminomethane-hydrochloric acid (Tris-hydrochloric acid) buffer having a pH of 7.6 and containing 0.2 M thorium chloride.

This rL-2 bath solution was poured into porous glass beads (chondral pore glass CPG-10, pore size 350A, 120-
200 mesh, Electro-Nucleoni
CS Co., Ltd.) column (column size is 16 diameter x
150 microliters and a capacity of 30 m/) were passed through the tube to adsorb 9-1 L-2. Note that this column of porous glass beads was prepared in advance with 0.4 M NaCl containing 0.4 M NaCl. IM l-lis-hydrochloric acid buffer (pH 7,6
) and was sufficiently equilibrated.

Then, 0.4 M NaClt (containing O, IMl.
200ml of lithium-hydrochloric acid buffer (pH 7,6) was passed through,
After washing the inside of the column, 2. O containing OM NaCl,
1Ml Lis-HCl buffer (+) H7,6) Buffer containing 1333% ethylene glycol (2,0M N, ic
0.1 M including l)! l Soluble hydrochloric acid buffer, pH 7
, 6) + 50 me to dissolve IL-2.

Dare The melt W curve is shown in Figure II.

After centrifuging for 30 minutes and removing the supernatant,
．． 05 M phosphate buffer (pH 7,0). I in salting out operation 1. , -2 showed almost no decrease in activity.

The obtained L-2 solution was subjected to gel filtration column chromatography once. As the resin, Sephaday 10-9y, G ] 00 (Sweden Pharma/
(manufactured by γ So), and the column used was 44Φ×9Q Cn+. Gel 4 (Q overcarano, flat NAi r, 1
．． 25-4 trichloride was used, and the same 1-2 buffer was used for rr- during elution. Flow rate 34me/hro IIl 5f for molecules is Ora
lbumin + Ribonuclease r increased.

Incidentally, the bathing curve of KEL dltI percolumn 1 column is shown in FIG.

The obtained Hill-I L-2 is Kelg percolumn If
f-z l.Cluffy (Sephadex G100) molecule iiL+ 5. 0 0 0 - 1 8. 0
0 0 'l) nF: ji'i- --- It is also possible to perform isoelectric focusing (Sweden).
Made by Pharmanoa Phase, 1131 =; 3 0 0 0
) P17.8 to 8.0 + 7.0 in Shikoyor analysis
Contains ingredients that cause +6.5. Furthermore, St.
) S-PAGE electrophoresis (Sweden Fart-Funan 1:i!J, GE-4 11)) The result is 14.000 and 16. O O O
It was shown to be composed of multiple components with a Dalton center, and did not contain any oleander proteins with molecular grooves larger than 25,000 Daltons.

The results of purification are shown in Table 113.

Table 1 In Table IV, the protein concentration was determined using the Protein Dye Kit manufactured by Bioracl, and the degree of purification was determined using the specific activity of the culture supernatant and the ratio of IL-2 solutions at each purification stage. This is the value obtained by dividing the activity.

Example 2 L-2 water-soluble I & (protein concentration 0.17m9) produced from JurkatX cells stimulated with Konkana...
/ go, specific activity 1'l 2 6 0 unit 7mg)
230 ml was adjusted to pH 7, 2V using 8mM potassium phosphate and sodium hydroxide, and then poured into a calanow filled with the same porous glass pieces used in Example 1.
(16mm x 35mm, bed volume 7ml
) A solution was passed through the tube to adsorb human l +--2. In addition, 0.02M phosphoric acid was added to the column layer. 4 [Equilibrated with J solution (pH 7.2).

After adsorbing IL-2, 0.02 M phosphate buffer (pH 7.2) was passed for 50 m, followed by a slow dilution of glinone-hydrochloric acid (pH 7.2) containing 0.2 M thorium chloride. pH 2.1) 50mg adsorbed l
L-2 was dissolved.

Purification degree 19 times, recovery rate 60%, specific activity 2500un i
t 1mg.

Example 3 Human II, L-2 production culture solution 215m obtained in the same manner as the human II,-2 production culture solution used in Example 1
l (protein concentration 0.68 mg/me, specific activity 3
6 2 units/-1 3 - my) was filled with the same porous J-lase heath as used in Example 2 (the size, bed volume, and equalization were the same as in Example e). . -2 was adsorbed, and 0.02 M phosphate buffer (pH 7.2)
After washing with 50 mg of resin, further washing with 50+++ l of 0.1 M + Lis-acetate buffer (pH 8, 0),
A part of the adsorbed 4''11 protein was eluted.

Next time 0. I M l-lys-acetic acid slow solution (pH
8.0) 11-2 was eluted with 30 ml of an eluent containing 75 M potassium thioanate.

Purification degree 14 times, recovery rate 80%, specific activity 1550 units
/mW.

Example 4 TI in human blood Hi1-1 produced from Tuba bulbs
1-2 solution (manufactured by Besescu Research Laboratories, protein concentration 0.82 / me, specific activity 122 units
/mg) 500 ml was equilibrated with 0.02 M phosphate buffer (p) + 7.2) Porous horn piece column (+6 mΦ
4+5me)-1 4-1 to adsorb human IL-2, 0.02 Mυ
acid buffer i & (pH 7,2) IOOme,
Furthermore, θ, 1M+-lys acetate buffer (pH 8,0) 10
Wash with O, IMl-IJ Soot-Acid Buffer containing 0.75 M thio/anoic acid) 50rn/!
Human IL-2 is eluted using this as an eluent.

Purification aid 13.5 times, recovery rate 559b.

Example 5 Commercially available →Norl L-2 solution (I TCGF-Ml 1 manufactured by Kikai Co., Ltd. 1-1 Antibody Research Institute, protein concentration 052)
m! // me, specific activity 250u old +7ml! )
175 ml was added to a porous glass bead column (+6 recessed ΦX4 (1 mm%
Pour the liquid through the tube (volume: 8 ml), and add 4 ml of liquid.
+--2 was adsorbed, followed by washing with 0.02 M phosphate buffer (pH 7,2) 5 Ome.

Next fl 2. Monkey IL-2 was eluted using 40 ml of an eluate containing 0.1 M Lis-HCl buffer (pH 8,0) containing OM NaCl and ethylene/glycol added to 33 ml.

Purification degree 88 times, recovery rate 90%.

Example 6 Commercially available Mouse+L-2 solution (manufactured by Hesestari@J-ThirahoraTries, protein concentration 0.78 mg/me, specific activity 12
B unit/mj7) 25 Ome was carried out (porous glass heath column equilibrated by the method described in E-112 (16 door Φ x 40 liver, bed volume 8 me)
0.02M'J acid buffer (pi17.2) 5
I washed it.

Next, 0.1 M Green-Hχ hydrochloric acid buffer (pl(2,2) 4 Ome) containing 0.5 M NaCl was passed through as an eluent to elute mouse IL-2.

Purification degree 17°5 times, recovery rate 75%.

Example 7 In the same manner as the culture solution used in Example 1, 1,000 me of the culture solution obtained in I~ (protein concentration 0.54 mg/me,
Specific activity: 11-450 u old t/my) Add solid ammonium sulfate 5967, strain to 85% of the saturated ammonium sulfate concentration, and add human I.
Proteins containing L-2 were precipitated. Other centrifugation 1mf
(6,000 rpm 30 minutes)
0mel was dissolved. This II, -2 solution was transferred to a dialysis tube (5 pectrum Medical 1n
dustires ilH 5pectrapor3
), add 0.01 M + Lys-HCl buffer (p
H7,6) 3 was dialyzed overnight in ionic strength, p
H was adjusted.

The obtained solution was equilibrated with the same buffer solution using a column of DEAE-Sepharose cL-6B (manufactured by Pharmanoa) (column size 16mm ΦXl 75mm, bed volume 35rn).
t) The solution was passed at a flow rate of t+5mel to adsorb human IL-2. Thereafter, after washing with the same buffer described in F.
．． 06 M +・') HCl buffer (pH 7,6)
Human IL-2 was eluted using lOOwl.

Purification degree 35 times, recovery rate 45%.

The first step is ion exchange-17.
- 100+++ g of crudely purified human 1 L-2 solution obtained by chromatography treatment and 100+++ g of solution with 2.339 thorium chloride added to make the NaCl concentration 0.4 M
Example 1 was equilibrated with 0.06 M) Lis-HCl buffer (p + - + 7.6) containing 0.4 M NaCl.
The column of porous glass beads mentioned above (+0.ΦX26
1 x 26 g 1 pef ml) was passed through the tube. After passing the solution,
0.06 M l-lith containing 0.4 M NaCl
Wash with 15+uJ of hydrochloric acid buffer (I) H7,6, and add 1
The plate was washed with 106M + Lis-HCl buffer (pH 7,6) containing 0.7M NaCl.

Then, human interleukin-2 was added to the eluent containing 1.5 M NaCl in O, 1 Ml Lys-HCl buffer (pi-18,0) F and 30% glycerin.
was eluted.

The degree of purification in the second step is 9.5 times, and the recovery rate is 75%.The degree of purification in the first and second steps is 332.5 times, and the recovery rate is 33%.

-18-

[Brief explanation of drawings]

Figure 1 shows the elution curve obtained by CPC; Color 11 chromatography.The solid line is the absorbance of the eluent at 280 nm f, and the dotted line is the eluent II--2? ; indicates divination. Figure 2 shows the elution curve of the gel-containing column Kuro'2 Togelafy, and the solid line is the absorbance of the cutting fluid at 280nrnt.
The dotted line indicates the l L -2 activity of the solution. Note that the l fraction is Il, 5 m/. Special 11 applicant Ajinomoto Co., Inc.-19=

Claims (1)

[Claims] (1) A solution containing INOCOLOIKI/・2 was brought into contact with a porous glass piece, and the INTERLOIGI/2 was sucked into the porous glass piece, and then the ink adsorbed from the porous glass piece with the eluent - ■】・IKIN 2 A method for purifying interleukin 2 that requires desorption and lysis. <・2) A solution containing inotaleugin 2 is prepared by adding 1 piece of porous member Karano, 1 piece of matogelaphy treatment, and gel Okako Karano. A method for purifying interleugin 2, which has unique characteristics compared to chromatography treatment.